Biological Fe-S clusters are nanoparticles containing 2-8 Fe atoms that are held together primarily by bridging S atoms. Proteins that contain Fe-S clusters serve a wide variety of essential tasks in living systems, including catalysis of chemical reactions, sensing the chemical environment, signaling to and repair of DNA, and the maintenance of molecular structure. Our main goals for this proposal center on four key questions: How do Fe-S clusters react with small molecules? How do they catalyze reactions, how do they work as nitric oxide and oxygen sensors, and what happens when they decompose? What are the steps in assembly of the hydrogenase active site `H-cluster'? How does the larger protein environment affect access of small molecules to Fe-S sites? How does chemistry at an Fe-S cluster affect tertiary protein structure and interactions with DNA? The expected outcomes from our research include: Information about the catalytic intermediates of enzymes that fix nitrogen (nitrogenase) or produce hydrogen (hydrogenase) Information about reaction intermediates when Fe-S cluster proteins react with signaling molecules NO and O2, and changes that occur as the cluster sensor transduces the signal to affect DNA Information about side chain contributions and gas channels in gas-processing enzymes that process or are inhibited by small molecules such as N2, O2, CO, and NO. The approach to gain this information is spectroscopy. Using photolysis/FT-IR, NRVS, and resonance Raman spectroscopy, this work will characterize how nitrogenase (N2ase) binds the inhibitor CO, as well as the structures of its complex with N2 and more reduced intermediates, elucidate when and how hydrides bound at the active sites of the hydrogenases (H2ases), Using time-resolved spectroscopies on a variety of time-scales, we aim to define intermediates and final products for reactions of NO and O2 with [4Fe-4S] clusters in various proteins, including the `WhiB' proteins from the tuberculosis-causing bacterium Mycobacterium tuberculosis. We will develop a new technique. 61Ni synchrotron Mssbauer spectroscopy, that should have broad applications to Ni enzymes and to chemistry in general.